Plant breeding depends on the efficient exploitation of genetic variation that resides in the germplasm of a particular crop species, and which determines the phenotype of a plant within a specific environment. While this is traditionally done by selection of a combination of desirable traits observed at the phenotypic level, this can increasingly be performed by selection on the basis of molecular markers which are genetically closely linked to the allelic form of a gene which contributes to the expression of a specific trait.
Selection of traits at the phenotypic level is complex for traits that manifest themselves in a recessive way. When one of the parents has one recessive and one dominant allele for a given gene, and the other parent has two dominant alleles for the same gene, the result of crossing of the two parents will be a population in which—in the absence of segregation distortion—half of the individuals carry one recessive allele for that gene. Those individuals can however not be recognised, since the desired recessive trait will not express itself in a heterozygous situation. Only after selfing and analyzing the offspring in the next generation can it be determined which individual was carrying the recessive allele, and hence which individual was heterozygous.
Usually a plant breeder or researcher wishes to proceed as fast as possible by identifying alleles of interest as early as possible in a plant population. In many situations, it is of particular interest to determine the genetic composition of a mother plant that has produced a particular seed. It is therefore an object of the present invention to provide an efficient and non-disruptive method for examining the genome of the maternal parent of a seed.
Techniques to analyze DNA are developing very rapidly, and they are becoming increasingly efficient and sensitive. These highly sensitive DNA analysis methods require only very small amounts of DNA to generate a reliable signal. The research leading to the present invention demonstrated that such low but detectable amounts of DNA are located on the seed coat surface.
A seed coat develops from degenerated ovule integuments, which is maternal tissue. The ploidy of the seed coat of a diploid plant is 2n, and genetically it is identical to the maternal parent of the seed, i.e. the parent on which the seed has originated and developed.
Various techniques exist for analyzing DNA from plants or seeds. They mainly differ from each other in the way biological material is collected, which tissues or plant organs are used therefor, and in which developmental stage sampling is performed. In commercial settings, it is advantageous to obtain genetic material for analysis (e.g. by means of molecular marker analysis or DNA-sequencing) without the necessity to grow the plants throughout their entire life cycle in order to produce seed of the next generation, or preferably even without the need to germinate the seeds and to grow plants altogether. Thus, one would like to collect DNA from a very early developmental stage, without jeopardising the survival and normal further development of the plantlets.
To traditionally obtain genetic information from a seed, either the seed has to be destroyed, damaged, or grown into a plant, after which the seed or plant tissue can be used for DNA extraction. The present invention, however, provides a non-disruptive and non-invasive method to obtain DNA from a seed coat, from which genetic information about the female parent of that seed can be gathered, without impairing the seed's capacity to germinate and to develop into a plant of the next generation.
Traditionally, seeds are germinated and grown into plantlets from which tissue is subsequently collected, at a developmental stage that allows the harvesting of a sufficiently large tissue fragment without causing critical damage to the plantlets. After DNA extraction, identification of the desired individuals takes place through the use of molecular markers. The remaining plants, which can represent a very large part of the sampled population, are identified as being undesired by the breeder for his targeted purpose, on the basis of their lack of one or more specific molecular markers. Such undesired plants will be discarded, as they merely occupy valuable space in the growth facilities.
An alternative method for obtaining DNA from a plantlet may comprise the collection of detached root border cells from germinated seeds (European patent application EP-1984496). Since no plant tissue is needed, the damage to the plantlets is essentially non-existing, and the speed and efficiency of this method is very high, while costs are low. However, this method still requires the germination of seeds in order to collect root border cells from a radicle or root, and this requires growth space.
Alternatively, DNA can be extracted from a seed, which can then be analysed to determine the genotype of the plant that the seed would give rise to, if the seed would be sown. The most common procedure to extract DNA involves crushing, grinding or destruction of the said seed in some other way, making it impossible to still use the particular plant individual that could have grown from that particular seed. An alternative method involves the cutting off of a small part of the seed, including the endosperm (but not including the embryo), by means of e.g. a cutting device or a laser beam, in such a way that the seed still remains viable and can be grown later on. Such methods are e.g. described in European patent application EP2200419, and in WO2011/082316, WO2011/119763, WO2011/163326 and WO2011/163362.
These techniques require sophisticated and complex equipment, since seeds need to be positioned in a precise and highly reproducible, consistent manner, and meticulously cut to not critically damage the embryo from which a plant should still develop after the tissue harvesting process. Either method takes a considerable amount of technical and experimental development, optimisation and testing time until reliable and useful results can potentially be achieved routinely for any given plant species.
A particular difficulty is the fact that even the seeds of a single species do not always have the exact same shape, size and weight, e.g. due to genetic and/or environmental variables, and this further complicates the practical application of such automated high-throughput seed chipping methods, even on seeds of a single species. Especially for species with round seeds such automated method is very difficult to standardise and optimise, because the exact position of the embryo inside seeds with such a shape is not predictable. Cutting off random parts of such seeds can easily lead to damage to the embryo and a high death rate among the sampled seeds, which is precisely what one wishes to avoid in a non-destructive method for selecting seeds with desired genotypic variants in order to subsequently grow them into plants.
Because of these differences in ploidy and origin, analysis of seed tissue can yield different results and can be used for various goals, depending on the type of tissue layer that is examined. Examination of a complete seed, or a part thereof containing the endosperm and/or embryo, results in information on the genotype of the plant it can generate. This genotype contains maternal as well as paternal genetic information. A study of a seed coat specifically yields data regarding the genome of the maternal parent from which the seed originates. However, it is technically very difficult to obtain DNA from the seed coat. Physical removal of the seed coat is a very tedious and precise activity, and it yields only a very small amount of tissue. Subsequently, the DNA needs to be extracted from the tissue, after which it can be assayed. Typically the seeds are strongly damaged by the removal of seed coat tissue, and they will usually not be usable anymore, certainly not for commercial purposes.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.